Pentachlorophenol mediated regulation of DAMPs and inflammation: In vitro study.

Pentachlorophenol (PCP) was once a widely employed organochlorine pesticide and wood preservative in United States. Due to its toxicity, the U.S. Environmental Protection Agency has classified it as a restricted-use pesticide and established as a liver carcinogen. Earlier reports have indicated increased production of inflammatory mediators like IL-1ß and TNF-α by immune cells, including NK cells, lymphocytes, or monocytes -on PCP exposure. Yet, there is only scant information available regarding the detailed molecular mechanisms affected by acute or chronic exposure of humans to PCP. Considering this, we examined PCP-induced inflammation and downstream signaling events in-(a) human lung adenocarcinoma cells (A549) with type II alveolar epithelial characteristics; and (b) human liver carcinoma cells (HepG2). Treatment of these cells with 1 µM and 10 µM concentration of PCP for 24 h duration resulted in a significant induction of cytokines/chemokines including IL-1ß, IL-6, TNF-α, IL-8, CCL2, and CCL5. Assessment of mRNA expression showed upregulated levels of danger-associated molecular patterns (DAMPs)-high mobility group box-1 (HMGB1) and heat shock protein 70 (Hsp70) as well as TLR-4 receptor in PCP-challenged cells. Increased expression of transcription factors-NF-κB and STAT3 provide further insight into the molecular mechanisms underlying PCP-induced toxicity/pathology. Interestingly, antibody-mediated neutralization of DAMPs abrogates PCP-mediated transcriptional induction of cytokines, chemokines and transcription factors in HepG2 and A549 cells. Overall, our findings demonstrate the important role of DAMPs in PCP-induced inflammatory responses.

Immunological and toxicological risk assessment of e-cigarettes.

Knowledge of the long-term toxicological and immunological effects of e-cigarette (e-cig) aerosols remains elusive due to the relatively short existence of vaping. Therefore, we performed a systematic search of articles published in public databases and analysed the research evidence in order to provide critical information regarding e-cig safety. Electronic nicotine delivery systems (or e-cigs) are an alternative to traditional cigarettes for the delivery of nicotine and are typically filled with glycerol or propylene glycol-based solutions known as e-liquids. Though present in lower quantities, e-cig aerosols are known to contain many of the harmful chemicals found in tobacco smoke. However, due to the paucity of experimental data and contradictory evidence, it is difficult to draw conclusive outcomes regarding toxicological, immunological and clinical impacts of e-cig aerosols. Excessive vaping has been reported to induce inflammatory responses including mitogen-activated protein kinase, Janus tyrosine kinase/signal transducer and activator of transcription and nuclear factor-κB signalling, similar to that induced by tobacco smoke. Based on recent evidence, prolonged exposure to some constituents of e-cig aerosols might result in respiratory complications such as asthma, chronic obstructive pulmonary disease and inflammation. Future studies are warranted that focus on establishing correlations between e-cig types, generations and e-liquid flavours and immunological and toxicological profiles to broaden our understanding about the effects of vaping.

Basic apoptotic and necrotic cell death in human liver carcinoma (HepG2 ) cells induced by synthetic azamacrocycle.

Treatment of diseases with synthetic materials has been an aspiration of mankind since the dawn of human development. In this research, three complex compounds of azamacrocycle (TD1, TD2, and TD3) were synthesized, and experiments were conducted to determine whether their toxicity to human liver carcinoma (HepG2 ) cells is associated with apoptotic and/or necrotic cell death. Cell survival was determined by MTT assay. Apoptosis and necrosis were measured by annexin V FITC/PI assay using the flow cytometry and by propidium iodide (PI) assay using the cellometer vision. HepG2 cells were treated with different concentrations of azamacrocycles for 48 h. Results from MTT assay indicated that all the three azamacrocycles significantly (p < 0.05) reduce cell viability in a dose-dependent manner, showing 48 h-LD50 values of about 37.97, 33.60, and 19.29 µM, for TD3, TD1 and TD2, respectively. Among the three compounds tested, TD2 showed the most pronounced cytotoxic activity against HepG2 cells, being about twofold more potent than TD3. The order of toxicity was TD2 > TD1 > TD3. Because TD2 exerted the most cytotoxic activity against HepG2 cells, it was used in the subsequent apoptosis and necrosis-related experiments. The flow cytometry assessment showed a strong dose-response relationship with regard to TD2 exposure and annexin V/PI positive cells. PI assay data indicated that TD2 exposure increased the proportion of fluorescence positive cells. Overall, our results indicate that azamacrocycle toxicity to HepG2 cells is associated with apoptotic and necrotic cell death resulting from phosphatidylserine externalization and loss of membrane integrity.

Neuregulin 1-Beta cytoprotective role in AML 12 mouse hepatocytes exposed to pentachlorophenol.

Neuregulins are a family of growth factor domain proteins that are structurally related to the epidermal growth factor. Accumulating evidence has shown that neuregulins have cyto- and neuroprotective properties in various cell types. In particular, the neuregulin-1 Beta (NRG1-Beta) isoform is well documented for its antiinflammatory properties in rat brain after acute stroke episodes. Pentachlorophenol (PCP) is an organochlorine compound that has been widely used as a biocide in several industrial, agricultural, and domestic applications. Previous investigations from our laboratory have demonstrated that PCP exerts both cytotoxic and mitogenic effects in human liver carcinoma (HepG2) cells, primary catfish hepatocytes and AML 12 mouse hepatocytes. We have also shown that in HepG2 cells, PCP has the ability to induce stress genes that may play a role in the molecular events leading to toxicity and tumorigenesis. In the present study, we hypothesize that NRG1-Beta will exert its cytoprotective effects in PCP-treated AML 12 mouse hepatocytes by its ability to suppress the toxic effects of PCP. To test this hypothesis, we performed the MTT-cell respiration assay to assess cell viability, and Western-blot analysis to assess stress-related proteins as a consequence of PCP exposure. Data obtained from 48 h-viability studies demonstrated a biphasic response; showing a dose-dependent increase in cell viability within the range of 0 to 3.87 microg/mL, and a gradual decrease within the concentration range of 7.75 to 31.0 microg/mL in concomitant treatments of NRG1-Beta+PCP and PCP. Cell viability percentages indicated that NRG1-Beta+PCPtreated cells were not significantly impaired, while PCP-treated cells were appreciably affected; suggesting that NRG1-Beta has the ability to suppress the toxic effects of PCP. Western Blot analysis demonstrated the potential of PCP to induce oxidative stress and inflammatory response (c-fos), growth arrest and DNA damage (GADD153), proteotoxic effects (HSP70), cell cycle arrest as consequence of DNA damage (p53), mitogenic response (cyclin- D1), and apoptosis (caspase-3). NRG1-Beta exposure attenuated stress-related protein expression in PCP-treated AML 12 mouse hepatocytes. Here we provide clear evidence that NRG1-Beta exerts cytoprotective effects in AML 12 mouse hepatocytes exposed to PCP.

Mitogenic and cytotoxic effects of pentachlorophenol to AML 12 mouse hepatocytes.

Pentachlorophenol (PCP), an organochlorine fungicide, is extensively used in the United States for the protection of wood products. Moreover, widespread agricultural, domestic, and industrial applications have caused PCP-contaminants to enter the food chain from the environment. There is accumulating evidence indicating that PCP is highly toxic to humans, and causes injury to major organs including the lung, liver, kidneys, heart, and brain. While PCP has been shown to induce systemic toxicity and carcinogenesis in several experimental studies, the literature is scarce regarding its toxic mechanisms of action. Recent investigations in our laboratory have shown that PCP exerts both cytotoxic and mitogenic effects in human liver carcinoma (HepG2) cells [1], and in primary culture of catfish hepatocytes [2]. In the present study, we hypothesized that PCP exposure will trigger similar cytotoxic and mitogenic responses in AML 12 Mouse hepatocytes. To test this hypothesis, we performed the MTT assay for cell viability in PCP-treated and control cells. Data obtained from this experiment indicated a biphasic response with respect to PCP toxicity; showing a hormosis effect characterized by mitogenicity at lower levels of exposure, and cytotoxicity at higher doses. Upon 48 hrs of exposure, PCP chemical doses required to cause 50% reduction in the viability (LC50) of AML 12 mouse hepatocytes was computed to be 16.0 + 2.0 microg/mL. These results indicate that, although the sensitivity to PCP toxicity varies from one cell line to another, its toxic mechanisms are similar across cell lines.

Pentachlorophenol-induced cytotoxic, mitogenic, and endocrine-disrupting activities in channel catfish, Ictalurus punctatus.

Pentachlorophenol (PCP) is an organochlorine compound that has been widely used as a biocide in several industrial, agricultural, and domestic applications. Although it has been shown to induce systemic toxicity and carcinogenesis in several experimental studies, the literature is scarce regarding its toxic mechanisms of action at the cellular and molecular levels. Recent investigations in our laboratory have shown that PCP induces cytotoxicity and transcriptionally activates stress genes in human liver carcinoma (HepG2) cells [1]. In this research, we hypothesize that environmental exposure to PCP may trigger cytotoxic, mitogenic, and endocrine-disrupting activities in aquatic organisms including fish. To test this hypothesis, we carried out in vitro cultures of male channel catfish hepatocytes, and performed the fluorescein diacetate assay (FDA) to assess for cell viability, and the Western Blot analysis to assess for vitellogenin expression following exposure to PCP. Data obtained from FDA experiments indicated a strong dose-response relationship with respect to PCP cytotoxicity. Upon 48 hrs of exposure, the chemical dose required to cause 50% reduction in cell viability (LD50) was computed to be 1,987.0 +/- 9.6 microg PCP/mL. The NOAEL and LOAEL were 62.5 +/- 10.3 microg PCP/mL and 125.0+/-15.2 microg PCP/mL, respectively. At lower levels of exposure, PCP was found to be mitogenic, showing a strong dose- and time-dependent response with regard to cell proliferation. Western Blot analysis demonstrated the potential of PCP to cause endocrine-disrupting activity, as evidenced by the up regulation of the 125-kDa vitellogenin protein the hepatocytes of male channel catfish.

Garlic attenuates hypercholesterolemic risk factors in olive oil fed rats and high cholesterol fed rats.

This study was undertaken to compare the effects of garlic (G) on hypercholesterolemic risk factors in rats fed with corn oil (C) or olive oil (O) with and without cholesterol (Ch) enrichment in the diet. Male Sprague-Dawley rats (n=6 per group) were fed semi-purified diets containing 5% oil with or without cholesterol (Ch) or garlic (G) for 21 days. In the C fed rats, addition of dietary Ch, there was a redistribution of Ch from HDL to LDL class. In contrast, 1% Ch added to O diets produced a 4-fold increase in serum Ch compared with levels in rats fed O without Ch. This was associated with a 14-fold increase in LDL Ch and a 7-fold decrease in HDL Ch levels. Addition of 2% G had no effect on the distribution of serum Ch between HDL and LDL Ch in C+Ch fed rats, but in O+Ch fed rats, G halved the increase in serum Ch and LDL and attenuated the decrease in serum HDL by 23%. The results suggest that the dietary O regulated the levels of serum in Ch loaded rats. G attenuated serum Ch in O fed rats and decreased serum risk factors, both total Ch and LDL-C ratio and LDL-Ch/HDL-Ch ratio.

CYP1a1, HSP70, P53, and c-fos expression in human liver carcinoma cells (HepG2) exposed to pentachlorophenol.

Pentachlorophenol (PCP) is a widely used biocidal compound with several industrial, agricultural and domestic applications. Although it has been shown to induce systemic toxicity and carcinogenesis in several experimental studies, the literature is scarce regarding its toxic mechanisms of action. Recent investigations in our laboratory have shown that PCP induces cytotoxicity and transcriptionally activates stress genes in human liver carcinoma (HepG2) cells [1]. We hypothesized that PCP-induced expression of stress proteins may play a role in the molecular events leading to toxicity and tumorigenesis in liver cells. To test this hypothesis, we performed the MTT-assay for cell viability, and the Western Blot and densitometric analyses to assess the expression of cellular protein including CYP1A1, c-fos, HSP70, and p53. Data obtained from the MTT-assay indicated a strong dose-relationship with respect to PCP cytotoxicity. The LD50 was computed to be 23.0 +/- 5.6 micrograms/mL. Western Blot and densitometric analyses also demonstrated a linear dose-response relationship with regard to CYP1A1 expression within the dose range of 0-50 micrograms/mL. However, a biphasic response was obtained with regard to HSP70, c-fos, and p53 expression, showing a peak induction at 25 micrograms/mL, and a drastic reduction in protein expression at 50 micrograms/mL, probably due to cell death at higher level of PCP exposure. At lower level of exposure, PCP was found to be mitogenic.